Process for the preparation of a mucosal form of the eosinophil cationic protein (ECP), mucosal ECP and uses thereof as allergic phlogosis marker

Abstract

It is described a process for the preparation of ECP from nasal mucosa of a subject; the ECP protein so obtainable; and a method for monitoring the phlogosis from allergic reaction of a subject.

1 . Process for the preparation of ECP from nasal mucosa of a subject, including the steps of: 
 submitting the subject to a provocation by means of an allergen suitable to induce the allergic phlogosis;    withdrawing after 24 hours from said subject nasal;    submitting said mucus to agitation and centrifugation in order to obtain a supernatant;    dissolving in said supernatant a chelating agent and concentrating the same from 5 to 30 times to obtain a concentrated sample;    submitting the concentrated sample to separation processes by molecular weight and/or ionic charge.    
     
     
         2 . Process for the preparation of ECP from nasal mucosa of a subject according to  claim 1  wherein the allergen suitable to induce the phlogosis is  Dermathophagoides pteronisinus.    
     
     
         3 . Process for the preparation of ECP from nasal mucosa of a subject according to  claim 1  wherein the first step is omitted and the nasal mucus withdrawn from subjects at the peak of a natural allergic phlogosis.  
     
     
         4 . Process for the preparation of ECP from nasal mucosa of a subject according to any one of the preceding claims wherein the chelating agent is EDTA.  
     
     
         5 . Process for the preparation of ECP from nasal mucosa of a subject according to any one of the preceding claims wherein the processes of separation by molecular weight include filtration through a Sephadex G-75 column.  
     
     
         6 . Process for the preparation of ECP from nasal mucosa of a subject according to any one of the preceding claims wherein the processes of separation by ionic charge include ion exchange chromatography through a CM-cellulose column.  
     
     
         7 . ECP protein obtainable according to the method of any one of the preceding claims from nasal mucus of subjects subjected to a natural or induced allergen provocation.  
     
     
         8 . Method for monitoring the phlogosis from allergic reaction of a subject, including the steps of: 
 incubating a biological fluid from the subject with an antibody obtained by immunization using the ECP protein according to  claim 7;     detecting the antigen-antibody reaction.    
     
     
         9 . Method according to  claim 8  wherein the detection of the antigen-antibody reaction is carried out using an additional antibody conjugated to a marker.